Polyketide-derived metabolites are found in both procaryote's (mainly actinomycetes) and eucaryotes where they display an amazing diversity of functional roles. Many of these compounds have important biological activities and have been developed as antibiotics and chemotherapeutic agents in both human and veterinary medicine. The importance of these compounds is increasing as the need arises to develop agents which effectively combat recalcitrant infectious diseases, and in the discovery of small molecules which mimic or inhibit the activity of natural hormones and immunomodulators. The primary aim of the proposed research is to understand the molecular mechanisms controlling carbon-chain construction in the biosynthesis of polyketide-derived macrolide antibiotics. Molecular genetic, biochemical and bioorganic chemical approaches will be used to obtain information on the functional role of each catalytic domain comprising the multifunctional (type I) polyketide synthase (PKS) including; condensing enzymes, acyl and terminal transferases, dehydrases, keto and enoylreductases, and acyl carrier proteins. Molecular genetic studies have revealed a close relationship between multifunctional fatty acid and polyketide synthases, and a model will be developed to dissect the mechanistic similarities and differences of these important biosynthetic systems. In order to establish an effective experimental model, we will investigate the methymycin (met) type I PKS of Streptomyces venezuelae. Although we expect the molecular genetic details will have important comparative value, this work will be pursued with the goal of establishing the S. venezuelae PKS as a novel system to study individual functional domains of macrolide-producing multifunctional PKSs. The versatility of the overall investigation is provided by the cross- disciplinary nature of the approach. Our initial efforts will include the overexpression and purification of the met multifunctional protein that mediates early biosynthetic steps including: carbon chain elongation, ketoreduction and dehydration. The ability to study these processes has only recently become possible due to technological advances that allow the overexpression of Streptomyces PKS proteins, and intact incorporation of chain elongation intermediates into macrolide antibiotics. Of primary importance is the mechanism of chain construction and functionalization of the nascent unsaturated fatty acid that is subsequently lactonized to form the macrolide molecule. Studies are designed to determine directly whether elaboration of the unsaturated fatty acid intermediate occurs linearly, as suggested by the deduced sequences of macrolide type I proteins. Structural analysis of the unsaturated fatty acids produced by in vitro conversion studies will allow us to analyze the precise functional contributions of individual PKS domains in the purified system. Overall, this work should provide important insight into the molecular genetic and biochemical relationships within type I PKS systems in Streptomyces and other actinomycetes. Moreover, it is hoped that this work will provide an important theoretical and experimental base for the rational production of novel macrolide metabolites using molecular genetic technology.